Mycobacterium tuberculosis (Mtb) causes pulmonary tuberculosis (TB) in humans, which was reported in 8.8 million people worldwide in 2011, leading to approximately 1.4 million deaths. This underscores the need for improved vaccine and novel antibiotics. We have identified two genes in Mtb (Rv3165c and Rv3167c) that are important for host cell death and autophagy inhibition and regulate resistance of Mtb to kanamycin. We hypothesize that these two genes are part of a bacterial signal transduction pathway that is engaged upon presence of Mtb in the phagosome and mediates a part of the transcriptional adaptation to the phagosome environment. Indeed, Rv3167c, encodes a protein with strong homology to the Tet repressor family of transcriptional regulators. In AIM1, we will use RNA-seq to simultaneously analyze the transcriptome of host and bacterium. We hypothesize that by comparing the results of wild-type Mtb infected cells to Rv3165c and Rv3167c deletion mutants we will be able to define the bacterial regulon controlled by the putative transcription factor Rv3167c. In addition, the impact of the Rv3167c regulon on the host cell transcriptional response will also be defined. We will identify the Rv3167c DNA-recognition site by ChIP-seq, determine if ligand binding to its C-terminal domain leads to activation or inhibition of binding to target DNA sites and characterize the Rv3165c and Rv3167c protein-protein interaction. Finally, we will analyze the roles of the Rv3167c regulated genes, Rv3168 and Rv3169, in mediating antibiotic resistance. In AIM2, we will use antibody arrays to characterize the differences in host cell signaling after infection with the two deletion mutants when compared to wild type Mtb. This will help to establish protein-signaling pathways important for cell death inhibition by Mtb and create great synergy with the RNS-seq analysis of AIM 1. In particular, we will explore the mechanisms of the Rv3167c Mtb deletion mutant induced host cell autophagy and programmed necrosis in ex vivo infection systems. Knock-out mice deficient in the identified key host cell signaling proteins will be used to corroborate the ex vivo findings during aerosol infection in vivo. Finally, wild-type and select knock-out mice strains will be used to investigate the in vivo virulence of the Rv3167c and Rv3165c mutants as determined by bacterial growth and survival of mice after aerosol infection. Overall, this proposal aims to characterize a novel Mtb signal transduction pathway and its importance for controlling host cell death and autophagy induction and bacterial resistance to antibiotics. We believe that the results we generate will have a significant impact on the development of novel immunotherapeutics and design of improved vaccines for treatment and prevention of this significant human disease.